This proposal will delineate the structural features of a group of Eph receptors (EphB2, EphA2, and EphA4) in their apo forms as well as in complex with their cognate ligands and peptides. These receptors are members of the largest receptor tyrosine kinase family identified to date, and have proven implications in both angiogenesis an tumorigenesis. The structure-function results of this proposal will provide a comprehensive description of the receptor/ligand interfaces across the family, including highly promiscuous receptors (EphB2, EphA4) and highly specific receptors (EphB4, EphA2). The combination of results from the structural analysis of the Eph receptor/ligand complexes and quantitative biophysical assays of Eph receptor-ligand binding will provide a set of structural determinants that can be used to optimize compound development for a specific Eph receptor. We have performed a high throughput screen with a mutant EphB4 receptor that was identified to show reduced affinity for the target ligand, the TNYL-RAW peptide. Nine compounds were identified form a small molecule compound repository at the NIH that selectively bind the EphB4 ligand binding domain. We will characterize all available compounds and compounds and compounds from similarity searches in cellular and structural assays. Given the important roles that EphB4, EphB2, EphA2 and EphA4 play in several disease areas including tumorigenesis, molecules specific for these receptors would form validated starting points for therapeutic compound development targeting processes involving protein-protein interactions such as angiogenesis and cell growth. Our studies will provide both the structural determinants and biophysical information that characterize the specific binding of these molecules to their target Eph receptors and will form a strong basis for further therapeutic development.